Detection antibody

Plasma concentrations of KIM-1, as well as tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2) as markers of chronic inflammation, were measured using a microbead-based assay as described previously.^{5 (link)} Samples were diluted ten-fold in sample diluent buffer (0.1M HEPES, 0.1M NaCl, 0.1% Tween-20 and 1% BSA; pH 7.4; filter sterilized), and 30 μL of diluted sample, recombinant standards and internal control samples were incubated with ~6000 microbeads that were coupled with KIM-1, TNFR1 and TNFR2 capture antibodies for 1 h (R&D Systems). After incubation, microbeads were washed 3X with PBST and incubated with corresponding detection antibodies (R&D Systems) for 45 min. After incubation, beads were washed 3X with PBS-Tween and incubated with Streptavidin-PE (Invitrogen) for 15 min. The signal from the fluorochrome, which is directly proportional to the amount of antigen bound at the microbead surface, was captured using the Bio-Plex system (Bio-Rad). The lower level of detection (LLOD) for KIM-1, TNFR1 and TNFR 2 were 1.02 pg/mL, 0.67 pg/ml, and 0.17 pg/ml, respectively and this value was assigned to all samples where analytes were not detected. As a marker of kidney function, we used creatinine mass spectrometry measurements^{12 (link)} conducted in the context of previous studies.^{11 (link)}